Despite intensive investigation, the mechanism of insulin action remains poorly understood. Much evidence suggests that as a consequence of insulin interaction with its cell surface receptor, the hormone initiates a series of reactions which coordinately modify the activity of a number of regulatory enzymes, controlling cellular metabolism. It appears that a critical intermediate step in this sequence of reactions involves an alteration in protein phosphorylation. We have shown that in adipose tissue and liver (using isolated rat adipocytes and hepatocytes) insulin can alter protein phosphorylation in at least two ways. First, insulin antagonizes CAMP-stimulated protein phosphorylation; second, insulin promotes the CAMP-independent phosphorylation of several cellular proteins. We have hypothesized that insulin achieves this latter effect by either of two mechanisms. 1) Insulin generates the accumulation of a unique, as yet unidentified, second messenger. 2) Insulin alters the concentration of certain ligands which interact with the substrates, and alter their properties as a substrate for kinase and/or phosphatases. In order to test these hypotheses, we have purified one of the substrates of insulin-stimulated phosphorylation, a hepatic 123,000 dalton 32P-peptide, and we have identified it as the enzyme, ATP-citrate lyase. This enzyme functions to provide the cytosolic acetyl CoA necessary for de novo fatty acid synthesis (lipogenesis), and we have shown that insulin stimulates lipogenesis in hepatocytes. We now wish to identify the effect of phosphorylation on the activity of ATP-citrate lyase. In addition, we will identify the site(s) of ATP-citrate lyase which are phosphorylated in response to insulin, and identify the protein kinases and phosphotases which act on these site(s). We will then examine whether insulin directly alters the activity of these enzymes, and if so, by which mechanism. Concomitantly, we plan to measure the change in various hepatic metabolite (ligand) concentrations in response to insulin, both in the cytosol and mitochondria. These studies will provide information not only on potential metabolite moderators of protein phosphorylation, but on the sites of hormonal control of lipogenesis as well, when coupled with appropriate measurements of enzyme activity.